Mutation is important for crop breeding and molecular genetic studies. Naturally arising bud mutation and artificial mutation are used. “Bud mutation” in the present invention indicates mutation that occurs in some somatic cells of a plant at the gene level. “Bud mutant” in the present invention indicates such a mutant. In the case of vegetatively propagated crops, such as flowers and ornamental plants and orchards, bud mutation that takes place without crossing provides genetic mutation important for developing varieties. However, the cause of the molecular mechanism of mutation due to bud mutation is unknown. Bud mutation depends on natural occurrence. Furthermore, natural mutation is thought to take place at a probability of only approximately one to one million. To increase such efficiency, radiation irradiation and techniques using chemical substances having mutagenicity have been used to induce mutation. When increase of a mutation occurrence rate is attempted, undesired mutation is introduced in addition to desired mutation. Thus, many cases result in obtainment of very poor phenotypes together with a target phenotype. This requires a search for a target mutant from among a mutant population comprising numerous plants and also results in low efficiency.
A transposon (also referred to as a transposable element) is a mobile genetic element that is broadly found among biological species ranging from prokaryotes to eukaryotes and that undergoes transposition on the genome. In plants, such transposon has been discovered in Arabidopsis thaliana, Brassica campestris, and the like [JP Patent Publication (Kokai) No. 2003-93074 A]. Furthermore, a transposon that controls the expression of an anthocyanin biosynthetic gene in maize (Zea mays) grains, snapdragon petals (Antirrhinum majus) [PROTEIN, NUCLEIC ACID AND ENZYME 37 1047-1059 (1992)], Japanese morning glory petals (Pharbitis nil) [Proc. Natl. Acad. Sci. U.S.A. 97: 7016-7023 (2000)], petunia petals (Petunia hybrida) [The Plant Journal 13: 39-50 (1998)], carnation petals (Dianthus caryophyllus), or the like is known well.
For example, in the case of a carnation variety having white petals with red spots, a transposon has been inserted within a dihydroflavonol-4-reductase (an anthocyanin biosynthetic enzyme hereinafter referred to as DFR) gene. The DFR gene is necessary for red pigment synthesis. When a transposon has been inserted, the DFR gene does not function, so that white flower color will be developed. However, it has been elucidated that the DFR gene function can be recovered and a pattern of red spots can be generated by excision of such inserted transposon at the time of petal formation [Plant Cell Physiology. 43: 578-585 (2002)]. Regarding the above-mentioned case of developing mutation sectors due to excision of a transposon in some cells within an individual plant, the involvement of a transposon has been elucidated in maize, snapdragon, Japanese morning glory, petunia, and the like via molecular genetics.
Furthermore, it has also been revealed that mutation due to a transposon is inherited via reproductive cells to progeny. In this case, the transposition of a transposon has taken place during the period of reproductive cell formation. Mutants resulting from such transposition can be obtained in the progeny. By the use of this phenomenon, a gene isolation technique has been developed that involves introducing a known transposon by gene transfer or the like and then analyzing mutants obtained in the progeny using the introduced transposon as a landmark. This technique is called transposon tagging [The Plant Journal 7: 677-685 (1995)].
As described above, the use of a transposon as a mutagen has been attempted. However, current techniques are insufficient for obtaining mutation due to the insertion of a transposon into a gene in buds. With such current techniques, mutants are obtained at best mainly via crossing or hybridization. Such case where the transposition of a transposon takes place among some cells composing an individual plant is known. For example, a case of bud mutation induced by a retrotransposon has been reported [Science 304: 982 (2004)]. However, no cases have been reported where such bud mutation that can be fixed by vegetative propagation is induced by a transposon other than retrotransposon.
Patent document 1 JP Patent Publication (Kokai) No. 2003-93074 A
Non patent document 1 PROTEIN, NUCLEIC ACID AND ENZYME 37 1047-1059 (1992)
Non patent document 2 Proc. Natl. Acad. Sci. U.S.A. 97: 7016-7023 (2000)
Non patent document 3 The Plant Journal 13: 39-50 (1998)
Non patent document 4 Plant Cell Physiology. 43: 578-585 (2002)
Non patent document 5 The Plant Journal 7: 677-685 (1995)
Non patent document 6 Science 304: 982 (2004)